In spite of abundant evidence from multiple laboratories that epigenetic and genetic develop during neoplastic evolution in Barrett's esophagus, relationships among genetic and epigenetic alterations are still poorly understood, in part because many studies investigate only one or the other and in part because few longitudinal studies have been performed. Project 1 will use novel and innovative methods to assess the evolution of clones with genetic and epigenetic alterations to determine the extent to which they predict neoplastic progression in a longitudinal cohort study of 614 patients with Barrett's esophagus (BE) with an anticipated 52,167 person-months of follow-up. We hypothesize that i) epigenetic abnormalities arise as early events in a limited number of CpG islands before widespread genomic instability;ii) early chromosomal instability in BE progression is characterized by localized regions of LOH and copy number change involving a relatively small number of genes (p16, p18INKc, PI3KR3) that in combination with early differential methylation of CpG islands in selected genes predispose to loss of TP53 and widespread chromosomal instability that develops as a late event in progression and iii) NSAID use modulates early evolution of genetic and epigenetic alternations in BE. Project 1will compare the sensitivity and specificity of a combined panel of epigenetic and genetic alterations to previously reported genetic (p16 LOH, TP53 LOH, tetraploidy, aneuploidy) and epigenetic (p16, RUNX3, HPP1) panels. Project 1 provides Project 2 with clonal genetic (LOH, copy number change and DNA content abnormalities) and epigenetic (differential methylation of CpG islands) biomarkers, as well as assessmentof clonal evolutionary dynamics to determine the genetic and epigenetic stages of progression that are most closely associated with host and environmental risk and protective factors. Project 1 also provides these measures to Project 3 to investigate the association of genetic instability biomakers (telomeres, fragile sites) with clonal evolution. Finally, Project 1 will develop clinically compatible DNA biomarker platforms for our validated markers so that they can be used in other centers and multicenter studies.